LPS-aggregating proteins GBP1 and GBP2 are each sufficient to enhance caspase-4 activation both in cellulo and in vitro.

The gamma-interferon (IFNγ)-inducible guanylate-binding proteins (GBPs) promote host defense against gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate sensing of the gram-negative bacterial outer membrane component lipopolysaccharide (LPS) by the noncanonical caspase-4 inflammasome. There are seven human GBP paralogs, and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS. The GBP1 microcapsule recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore gram-negative-induced pyroptosis in GBP1KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates "free" LPS through protein polymerization. We demonstrate that supplementation of either recombinant polymerized GBP1 or GBP2 to an in vitro reaction is sufficient to enhance LPS-induced caspase-4 activation. This provides a revised mechanistic framework for noncanonical inflammasome activation where GBP1 or GBP2 assembles cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to gram-negative bacterial infections.

The bacterial effector GarD shields Chlamydia trachomatis inclusions from RNF213-mediated ubiquitylation and destruction.

Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infections and a major threat to women's reproductive health in particular. This obligate intracellular pathogen resides and replicates within a cellular compartment termed an inclusion, where it is sheltered by unknown mechanisms from gamma-interferon (IFNγ)-induced cell-autonomous host immunity. Through a genetic screen, we uncovered the Chlamydia inclusion membrane protein gamma resistance determinant (GarD) as a bacterial factor protecting inclusions from cell-autonomous immunity. In IFNγ-primed human cells, inclusions formed by garD loss-of-function mutants become decorated with linear ubiquitin and are eliminated. Leveraging cellular genome-wide association data, we identified the ubiquitin E3 ligase RNF213 as a candidate anti-Chlamydia protein. We demonstrate that IFNγ-inducible RNF213 facilitates the ubiquitylation and destruction of GarD-deficient inclusions. Furthermore, we show that GarD operates as a cis-acting stealth factor barring RNF213 from targeting inclusions, thus functionally defining GarD as an RNF213 antagonist essential for chlamydial growth during IFNγ-stimulated immunity.

Interferon-Inducible E3 Ligase RNF213 Facilitates Host-Protective Linear and K63-Linked Ubiquitylation of Toxoplasma gondii Parasitophorous Vacuoles.

The obligate intracellular protozoan pathogen Toxoplasma gondii infects a wide range of vertebrate hosts and frequently causes zoonotic infections in humans. Whereas infected immunocompetent individuals typically remain asymptomatic, toxoplasmosis in immunocompromised individuals can manifest as a severe, potentially lethal disease, and congenital Toxoplasma infections are associated with adverse pregnancy outcomes. The protective immune response of healthy individuals involves the production of lymphocyte-derived cytokines such as interferon gamma (IFN-γ), which elicits cell-autonomous immunity in host cells. IFN-γ-inducible antiparasitic defense programs comprise nutritional immunity, the production of noxious gases, and the ubiquitylation of the Toxoplasma-containing parasitophorous vacuole (PV). PV ubiquitylation prompts the recruitment of host defense proteins to the PV and the consequential execution of antimicrobial effector programs, which reduce parasitic burden. However, the ubiquitin E3 ligase orchestrating these events has remained unknown. Here, we demonstrate that the IFN-γ-inducible E3 ligase RNF213 translocates to Toxoplasma PVs and facilitates PV ubiquitylation in human cells. Toxoplasma PVs become decorated with linear and K63-linked ubiquitin and recruit ubiquitin adaptor proteins in a process that is RNF213 dependent but independent of the linear ubiquitin chain assembly complex (LUBAC). IFN-γ priming fails to restrict Toxoplasma growth in cells lacking RNF213 expression, thus identifying RNF213 as a potent executioner of ubiquitylation-driven antiparasitic host defense. IMPORTANCE Globally, approximately one out of three people become infected with the obligate intracellular parasite Toxoplasma. These infections are typically asymptomatic but can cause severe disease and mortality in immunocompromised individuals. Infections can also be passed on from mother to fetus during pregnancy, potentially causing miscarriage or stillbirth. Therefore, toxoplasmosis constitutes a substantial public health burden. A better understanding of mechanisms by which healthy individuals control Toxoplasma infections could provide roadmaps toward novel therapies for vulnerable groups. Our work reveals a fundamental mechanism controlling intracellular Toxoplasma infections. Cytokines produced during Toxoplasma infections instruct human cells to produce the enzyme RNF213. We find that RNF213 labels intracellular vacuoles containing Toxoplasma with the small protein ubiquitin, which functions as an "eat-me" signal, attracting antimicrobial defense programs to fight off infection. Our work therefore identified a novel antiparasitic protein orchestrating a central aspect of the human immune response to Toxoplasma.

Detection of Cytosolic Shigella flexneri via a C-Terminal Triple-Arginine Motif of GBP1 Inhibits Actin-Based Motility.

Dynamin-like guanylate binding proteins (GBPs) are gamma interferon (IFN-γ)-inducible host defense proteins that can associate with cytosol-invading bacterial pathogens. Mouse GBPs promote the lytic destruction of targeted bacteria in the host cell cytosol, but the antimicrobial function of human GBPs and the mechanism by which these proteins associate with cytosolic bacteria are poorly understood. Here, we demonstrate that human GBP1 is unique among the seven human GBP paralogs in its ability to associate with at least two cytosolic Gram-negative bacteria, Burkholderia thailandensis and Shigella flexneri Rough lipopolysaccharide (LPS) mutants of S. flexneri colocalize with GBP1 less frequently than wild-type S. flexneri does, suggesting that host recognition of O antigen promotes GBP1 targeting to Gram-negative bacteria. The targeting of GBP1 to cytosolic bacteria, via a unique triple-arginine motif present in its C terminus, promotes the corecruitment of four additional GBP paralogs (GBP2, GBP3, GBP4, and GBP6). GBP1-decorated Shigella organisms replicate but fail to form actin tails, leading to their intracellular aggregation. Consequentially, the wild type but not the triple-arginine GBP1 mutant restricts S. flexneri cell-to-cell spread. Furthermore, human-adapted S. flexneri, through the action of one its secreted effectors, IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis These studies reveal that human GBP1 uniquely functions as an intracellular "glue trap," inhibiting the cytosolic movement of normally actin-propelled Gram-negative bacteria. In response to this powerful human defense program, S. flexneri has evolved an effective counterdefense to restrict GBP1 recruitment.IMPORTANCE Several pathogenic bacterial species evolved to invade, reside in, and replicate inside the cytosol of their host cells. One adaptation common to most cytosolic bacterial pathogens is the ability to coopt the host's actin polymerization machinery in order to generate force for intracellular movement. This actin-based motility enables Gram-negative bacteria, such as Shigella species, to propel themselves into neighboring cells, thereby spreading from host cell to host cell without exiting the intracellular environment. Here, we show that the human protein GBP1 acts as a cytosolic "glue trap," capturing cytosolic Gram-negative bacteria through a unique protein motif and preventing disseminated infections in cell culture models. To escape from this GBP1-mediated host defense, Shigella employs a virulence factor that prevents or dislodges the association of GBP1 with cytosolic bacteria. Thus, therapeutic strategies to restore GBP1 binding to Shigella may lead to novel treatment options for shigellosis in the future.

Microcins mediate competition among Enterobacteriaceae in the inflamed gut.

The Enterobacteriaceae are a family of Gram-negative bacteria that include commensal organisms as well as primary and opportunistic pathogens that are among the leading causes of morbidity and mortality worldwide. Although Enterobacteriaceae often comprise less than 1% of a healthy intestine's microbiota, some of these organisms can bloom in the inflamed gut; expansion of enterobacteria is a hallmark of microbial imbalance known as dysbiosis. Microcins are small secreted proteins that possess antimicrobial activity in vitro, but whose role in vivo has been unclear. Here we demonstrate that microcins enable the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to limit the expansion of competing Enterobacteriaceae (including pathogens and pathobionts) during intestinal inflammation. Microcin-producing EcN limits the growth of competitors in the inflamed intestine, including commensal E. coli, adherent-invasive E. coli and the related pathogen Salmonella enterica. Moreover, only therapeutic administration of the wild-type, microcin-producing EcN to mice previously infected with S. enterica substantially reduced intestinal colonization by the pathogen. Our work provides the first evidence that microcins mediate inter- and intraspecies competition among the Enterobacteriaceae in the inflamed gut. Moreover, we show that microcins can act as narrow-spectrum therapeutics to inhibit enteric pathogens and reduce enterobacterial blooms.

The number of sentinel nodes identified as prognostic factor in oral epidermoid cancer.

In patients with oral cavity epidermoid cancer without palpable nodes, the standard cervical treatment is supraomohyoid dissection; nevertheless, lymphatic mapping with sentinel node (SN) biopsy has been useful and allows the identification of a group of patients where neck dissection may be prevented. The objective of this study was to examine which factors diagnose the possibility of metastasis in SN. A non-randomized prospective study was performed during a two year period and included patients with T1-2, N0 oral cavity cancer >4 mm thick, SN was identified by blue dye and Rhenium colloid. All patients underwent pre-operatory lymphogammagraphy and elective-selective neck dissection. Age, gender, tumor site, T, tumor thickness and number of sentinel nodes found were evaluated. In 48 patients, at least one sentinel node was found, 2 per patient on average. 10.4% showed drainage outside the supraomohyoid region, 13/48 had nodular metastasis, 4 with negative SN(8.3%). T, location, thickness and number of SN are related to non-SN metastasis. Identification of 3SN or more was related to the possibility of SN metastasis p=0 (RR 10.1, I. C. 95% 1.1 91.2). The combined technique (dye-colloid) offers a high index of success in the identification of SN in patients with oral cavity cancer, while lymphogammagraphy identifies patients with anatomically unexpected drainage. Patients with T1 less than 2cm, not located on the tongue with thickness <5 mm and more than 2 SN were less likely to have metastasis in non-sentinel nodes. The identification of at least 3 sentinel nodes decreases the possibility of identifying patients with hidden metastasis (p=0.04).